Replaceable Parylene Membranes For Nanocalorimeter

ABSTRACT

A measurement operation, such as a calorimetry measurement, is performed using a measurement array and a replaceable passivation membrane (e.g., a parylene membrane). The passivation membrane is used to cover the measurement array to provide temporary electrical and chemical passivation, while still allowing measurement of the parameter of interest (e.g., temperature, in a calorimetry measurement). By only replacing the membrane instead of the entire measurement array between measurement operations, the cost of the measurements can be significantly reduced over conventional methods. The passivation membrane can be mounted on a frame to simplify handling of the membrane.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/741,635, entitled “Replaceable Parylene Membranes ForNanocalorimeter” filed Dec. 19, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of calorimetry, and in particular, toa system and method for reducing nanocalorimetry operating costs.

2. Related Art

Calorimetry is used to measure enthalpic changes, including enthalpicchanges arising from reactions, phase changes, changes in molecularconformation, temperature variations, and other variations of interestthat may occur for a particular specimen. By measuring enthalpic changesover a series of conditions, other thermodynamic variables may bededuced.

For example, measurements of enthalpy as a function of temperaturereveal the heat capacity of a specimen, and titrations of reactingcomponents can be used to deduce the binding constant and effectivestoichiometry for a reaction. Calorimetry measurements are useful in abroad variety of applications, including, for example, pharmaceuticals(drug discovery, decomposition reactions, crystallization measurements),biology (cell metabolism, drug interactions, fermentation,photosynthesis), catalysts (biological, organic, or inorganic),electrochemical reactions (such as in batteries or fuel cells), andpolymer synthesis and characterization, to name a few.

In general, calorimetry measurements can be useful in the discovery anddevelopment of new chemicals and materials of many types, as well as inthe monitoring of chemical processes. Standard calorimeters requirerelatively large samples (typically about 0.5 ml to 10 liters) andusually measure one sample at a time. As such, these systems cannot beused to measure very small samples, as might be desired for precious orhighly reactive materials. Furthermore, standard calorimeters cannot beused effectively to monitor a large number of reactions of small samplesize in parallel, as is required in order to perform studies usingcombinatorial chemistry techniques.

In recent years, researchers and companies have turned to combinatorialmethods and techniques for discovering and developing new compounds,materials, and chemistries. For example, pharmaceutical researchers haveturned to combinatorial libraries as sources of new lead compounds fordrug discovery. As another example, Symyx Technologies™ is applyingcombinatorial techniques to materials discovery in the life sciences,chemical, and electronics industries.

Consequently, there is a need for tools that can measure reactions andinteractions of large numbers of very small samples in parallel,consistent with the needs of combinatorial discovery techniques.Preferably, users desire that these tools enable inexpensivemeasurements and minimize contamination and cross-contaminationproblems.

One of the most popular uses of combinatorial techniques to date hasbeen in pharmaceutical research. Pharmaceutical researchers have turnedto combinatorial libraries as sources of new lead compounds for drugdiscovery. A combinatorial library is a collection of chemical compoundsthat have been generated, by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” asreagents. For example, a combinatorial polypeptide library is formed bycombining a set of amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can theoretically besynthesized through such combinatorial mixing of chemical buildingblocks.

Once a library has been constructed, it must be screened to identifycompounds, which possess some kind of biological or pharmacologicalactivity. For example, screening can be done with a specific biologicalcompound, often referred to as a target, that participates in a knownbiological pathway or is involved in some regulation function. Thelibrary compounds that are found to react with the targets arecandidates for affecting the biological activity of the target, andhence a candidate for a therapeutic agent.

Through the years, the pharmaceutical industry has increasingly reliedon high throughput screening (HTS) of libraries of chemical compounds tofind drug candidates. HTS describes a method where many discretecompounds are tested in parallel so that large numbers of test compoundsare screened for biological activity simultaneously or nearlysimultaneously. Currently, the most widely established techniquesutilize 96-well microtitre plates. In this format, 96 independent testsare performed simultaneously on a single 8 cm by 12 cm plastic platethat contains 96 reaction wells. These wells typically require assayvolumes that range from 50 to 500 microliters. In addition to theplates, many instruments, materials, pipettors, robotics, plate washersand plate readers are commercially available to fit the 96-well formatto a wide range of homogeneous and heterogeneous assays. To achievefaster testing, the industry is evolving to plates that contain 384 and1536 wells.

A variety of measurement approaches has been used to screencombinatorial libraries for lead compounds, one of which is theinhibitor assay. In the inhibitor assay, a marker ligand, often thenatural ligand in a biological pathway, is identified that will bindwell with the target protein molecule. The assay requires the chemicalattachment of a fluorescent molecule to this marker ligand such that thefluorescent molecule does not affect the manner in which the markerligand reacts with the target protein. To operate an inhibitor assay,the target protein is exposed to the test ligands in microtitre wells.After a time necessary for reaction of the test ligand to the targetprotein, the marker ligand is applied. After a time for reaction withthe marker ligand, the wells are rinsed such that non-reacted markerligand is washed away. In wells where the target protein and the testligand have reacted, the test ligand blocks the active site of thetarget protein so the marker ligand cannot react and is washed away,while in cells where the target protein and test ligand have notreacted, the marker ligand reacts with the target protein and is notwashed away. By investigating the wells for the presence of fluorescenceafter the washing, reactions of test ligands and target proteins can bedetermined as having occurred in wells where no fluorescence isobservable.

However, the inhibitor assay requires time and expense to develop theassay. The principal components that need development are discovering amarker ligand and attaching a fluorophore to the marker in a manner thatdoes not affect its reaction with the target protein. Attaching thefluorescent marker can often take 3 months of development or more andcost $250 k or more once the marker ligand is identified. An assaymethod that avoids such assay development, such as measuring the heat ofthe reaction with calorimetry, would eliminate this cost and lime delayin the discovery process.

Calorimetry measurements are commonly utilized in biophysical andbiochemical studies to determine energy changes as indications ofbiochemical reactions in a media. Prior techniques for measurementsinclude using electrodes, thermopiles, optical techniques, andmicrocalorimeters for measurements within a sampled media. There is agreat interest in developing calorimetry devices, and in particular,ultra-miniature microcalorimeter devices (i.e., nanocalorimetrydevices), that require very small volumes of sampled media and that canquickly measure large numbers of reactions. Ideally, those reactionmeasurements can provide efficient assays; e.g., inhibitor assays whichcan be used in HTS to screen roughly 100,000 test ligands a day.

Accordingly modern calorimetry tools (in particular, microcalorimetryand nanocalorimetry tools) include an array of detectors that allowmultiple measurement operations to be performed simultaneously. FIG. 1Ashows a top view of a nanocalorimeter array 100, which is similar tonanocalorimeter arrays described in detail in co-owned, co-pending U.S.patent application Ser. No. 2003/0186453, herein incorporated byreference.

Nanocalorimeter array 100 includes a frame 110 and two detectors 120.Detectors 120 are commonly amorphous silicon (a-Si) structures that areformed on a Kapton plastic film (shown in FIG. 1B), which in turn issupported by frame 110. Detectors 120 include the devices necessary toperform calorimetry measurements on sample droplets 190 of testmaterial. The measurement data is then read out from detectors 120 viacontacts 111 on the periphery of frame 110.

In addition, microcalorimeter and nanocalorimeter arrays, such asnanocalorimeter array 100, typically include a thin (1-3 μm) parylenecoating 130 that is deposited over detectors 120 (and frame 110). Sampledroplets 190 are placed directly onto parylene coating 130, whichprovides a hydrophobic surface that facilitates the merging and mixingof sample droplets 190. Parylene coating 130 also provides electricaland chemical passivation for detectors 120, while still allowing thethermal effects of droplet interactions to be measured by detectors 120.

The coverage provided by parylene coating 130 is more clearly depictedin FIG. 1B, which shows a cross section of nanocalorimeter array 100. Asdescribed above with respect to FIG. 1A, parylene coating 130 coversdetectors 130, which in turn are formed on Kapton™ layer 131 (copperstrips 132 beneath detectors 130 are isothermal elements to ensure thatthe measurement elements of detectors 120 are thermally coupled tosample droplets 190).

To avoid contamination, nanocalorimeter arrays (such as nanocalorimeterarray 100) are typically discarded after a single use, which can addsignificant costs to large nanocalorimetry experiments. While parylenecoating 130 can sometimes be cleaned and sterilized to enable re-use ofnanocalorimeter array 100, such refurbishment activity can betime-consuming and expensive.

Accordingly, it is desirable to provide a system and method forperforming calorimetry operations that enables reuse of calorimeterarrays.

SUMMARY OF THE INVENTION

By using a replaceable (removable) passivation membrane, the inventionbeneficially provides a simple means by which calorimeter arrays can bere-used. According to an embodiment of the invention, the passivationmembrane is used to cover the uncoated measurement elements of acalorimeter array. Sample droplets are deposited onto the passivationmembrane, and the calorimetry measurements are taken. Once themeasurements are completed, the passivation membrane is simply removedfrom the calorimeter array. A new passivation membrane can then beplaced over the calorimeter array to allow the next measurementoperation to be performed.

Because only the passivation membrane is replaced for each newmeasurement operation, the invention can significantly reduce the totalcost of a series of measurement operations compared to conventionalsingle-use calorimeter arrays. According to an embodiment of theinvention, the passivation membrane can be mounted on a rigid frame tofacilitate placement of the passivation membrane over the calorimeterarray, thereby improving measurement throughput by simplifying thereplacement of the passivation membrane after each measurementoperation.

According to an embodiment of the invention, the passivation membranecan be produced by depositing a thin layer of the passivation materialonto a form created by a frame and a temporary backing structure mountedto the frame. A thin layer of the desired membrane material is thendeposited on the exposed portions of the frame and the backing structurewithin the frame. The membrane layer is then released from the backingstructure (via a low-adhesion film on the backing structure), leavingthe membrane supported by only the frame.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a top view of a nanocalorimeter.

FIG. 1B is a cross section of the nanocalorimeter of FIG. 1A.

FIGS. 2A and 2B depict a measurement system in accordance with anembodiment of the invention.

FIG. 3 is a flow diagram of a calorimetry operation performed inaccordance with an embodiment of the invention.

FIGS. 4A, 4B, and 4C depict a method for forming a disposablepassivation membrane, in accordance with an embodiment of the invention.

DETAILED DESCRIPTION

FIG. 2A shows a measurement system 201 that includes a replaceablepassivating membrane 282, in accordance with an embodiment of theinvention. Measurement system 201 includes an array mount 202 forsupporting a measurement array 200, a membrane mount 203 for supportinga membrane assembly 280, and an actuator mechanism 204 for moving arraymount 202 and membrane mount 203 towards and away from each other.

Measurement array 200 includes multiple detectors 220 formed on asupport layer 231, which is in turn supported by a frame 210. Supportlayer 231 can comprise any material that can maintain detectors 220 in asubstantially fixed position relative to frame 210. For example,according to an embodiment of the invention, support layer 210 cancomprise a polyimide (e.g., Kapton™) film.

Detectors 220 provide the desired measurement capabilities formeasurement array 200. For example, if measurement array 200 comprises acalorimeter array, detectors 220 can comprise thermal sensors for heatmeasurements. Alternatively, if measurement array 200 comprises aphotometric array, detectors 220 can comprise photosensors for radiationmeasurements.

Note that while two detectors 220 are depicted for exemplary purposes,measurement array 200 can comprise any number of detectors 220.Measurement data from detectors 220 are read out via contacts 211 at theperiphery of measurement array 200.

Note further that, for exemplary purposes, measurement array 200 isdepicted to be substantially similar to nanocalorimeter array 100 shownin FIGS. 1A and 1B, except that measurement array 200 does not includethe integral parylene coating 180 of nanocalorimeter array 100. However,as noted above, measurement array 200 can comprise any type ofmeasurement structure.

Passivating membrane 282 is incorporated into a membrane assembly 280that also includes a support frame 281. Support frame 281 provides arigid structure that supports the perimeter of passivating membrane 282,thereby maintaining passivating membrane 282 in a substantially planarconfiguration.

Passivating membrane 282 itself can comprise any flexible material thatprovides sufficient electrical and chemical passivation against thesample liquids to being measured, while still allowing proper transferof the measurement parameter of interest. For example, according to anembodiment of the invention, passivating membrane 282 can be a 1-3 μmthick parylene membrane for nanocalorimetry measurements.

In FIG. 2A, membrane mount 203 holds membrane assembly 280 abovemeasurement array 200, so that passivation membrane 282 is facingdetectors 220. Then, in FIG. 2B, actuator 204 moves membrane mount 203towards array mount 202 (as indicated by the arrow). This pressesmeasurement array 200 into passivation membrane 282, so that membrane282 covers detectors 220 (e.g., stretched over detectors 220). In thismanner, membrane 282 effectively becomes a temporary passivation layerfor measurement array 200.

Consequently, when sample droplets 290 are subsequently deposited ontopassivating membrane 282, detectors 220 are chemically and electricallyisolated from those sample droplets 290. Meanwhile, the thermalcharacteristics of sample drops 290 (e.g., heat of reaction) aretransmitted through passivating membrane 220 and can therefore bemeasured by detectors 220. The measurement data from detectors 220 canthen be read out from contacts 211.

According to an embodiment of the invention, a preformed contact opening204 can be included in passivating membrane 282 at the location of eachcontact 211 to provide electrical access. According to anotherembodiment of the invention, a contact pin 205 can be used at eachcontact 211 to pierce passivating membrane 282. According to anotherembodiment of the invention, electrical connectivity can be achievedthrough the edges or backside of frame 210.

According to an embodiment of the invention, measurement array 200 canhave a slightly “bowed” (convex) configuration, such that the firstcontact with passivating membrane 282 is made towards the center ofmeasurement array 200. The contact area between passivating membrane 282and measurement array 200 will then propagate outward to minimize thechances of air pocket formation between passivating membrane 282 andmeasurement array 200.

Once the calorimetry measurement operation is complete, actuator 204 canmove membrane mount 203 away from array mount 202, thereby liftingpassivating membrane 282 off of measurement array 200. The used membraneassembly 280 in membrane mount 203 can then be replaced with a newmembrane assembly, and another measurement operation can immediately beperformed by measurement system 201.

In this manner, multiple measurement operations can be performed inrapid succession. In addition, because the expensive measurement array200 can be re-used, the cost of those measurements can be significantlyreduced over conventional (single use) systems. Furthermore, theprecision and capabilities of measurement array 200 can be increased,since the higher cost associated with those performance enhancements canbe offset by the fact that measurement array 200 can be re-used.

FIG. 3 shows a flow chart depicting a calorimetry measurement process inaccordance with an embodiment of the invention (such as described withrespect to FIGS. 2A and 2B). In a “COVER ARRAY WITH (NEW) MEMBRANE” step310, a passivating membrane (e.g., passivating membrane 280) is used tocover the detectors (e.g., detectors 220) of a measurement array (e.g.,measurement array 200). Then, when sample droplets (e.g., sampledroplets 290) are placed on the passivating membrane in a “DEPOSITSAMPLE DROPLET(S) ONTO MEMBRANE” step 320, the passivating membraneprevents unwanted chemical and/or electrical interactions between thosesample droplets and the detectors of the measurement array.

At the same time, because the passivating membrane is pressed tightlyagainst the detectors of the calorimeter array, measurements (e.g.,enthalpic or photometric measurements) can be taken by the detectors ina “TAKE MEASUREMENTS USING ARRAY” step 330. Once the measurementoperation complete, the passivating membrane is removed from themeasurement array in a “LIFT MEMBRANE OFF ARRAY” step 340. If anothermeasurement is to be taken, the process loops back to step 310, where anew passivating membrane is placed over the measurement array.

As described with respect to FIGS. 2A and 2B, the passivating membranecan be pre-mounted on a frame (membrane assembly 280) to simplify thestep of placing the membrane onto the calorimeter array (step 310).FIGS. 4A-4C depict a method for creating such a mounted membrane, inaccordance with an embodiment of the invention. In FIG. 4A, a backingstructure 486 is clamped on to a membrane frame 281. Backing structureincludes a planar region 487 that is enclosed by frame 281.

Planar region 487 is formed by a low-adhesion film 485, which can be anymaterial that can be readily separated from the desired passivationmembrane material. For example, according to various embodiments of theinvention, low-adhesion film 485 can be a non-stick surface (e.g.,Teflon™), a thermal or UV (ultra-violet) release tape (e.g., Revalpha™from Nitto Denko Corp.), or even a sacrificial layer that can bedissolved by an appropriate etchant.

In FIG. 4B, a flexible layer 282′ of the desired membrane material isformed over planar region 487 and the portion of membrane frame 281 thatsurrounds planar region 487. The specific means by which flexible layer282′ is formed will depend on the desired membrane material. Forexample, according to an embodiment of the invention, flexible layer282′ can be created by vapor-depositing parylene over planar region 487and the surrounding portions of frame 281.

Finally, in FIG. 4C, flexible layer 282′ and membrane frame 281 arereleased from backing structure 486 to create a membrane assembly 280 inwhich passivating membrane 282 is supported by frame 281. As notedabove, this release operation can be performed in a variety of ways,depending on the nature of low-adhesion film 485. According to anembodiment of the invention, a mechanical peel operation can be used toseparate membrane 282 from low-adhesion film 485. According to anotherembodiment of the invention, thermal or UV radiation could be used toreduce the adhesion provided by low-adhesion film 485. According toanother embodiment of the invention, a gas or liquid etchant could beused to dissolve low-adhesion film 485.

Although the present invention has been described in connection withseveral embodiments, it is understood that this invention is not limitedto the embodiments disclosed, but is capable of various modificationsthat would be apparent to one of ordinary skill in the art. For example,rather than forming a membrane via vapor deposition (as described withrespect to FIGS. 4A-4C), membrane assembly 280 could be created byaffixing a pre-existing sheet of the desired material to a frame.Furthermore, passivation membrane 282 could be placed over measurementarray 200 without the use of a membrane frame 281; e.g., by providing aroll of thin parylene sheet and unrolling an appropriate portion over acalorimeter array. In addition, the replaceable passivation layer can beused with any type of measurement array that requires chemical and/orelectrical passivation. For example, a clear passivation membrane couldbe used with a photometric array to provide environmental protectionwithout interfering with the operation of the light sensors. Therefore,the invention is limited only by the following claims and theirequivalents.

1. A method for creating membrane assembly, the membrane assemblycomprising a membrane of a first material supported by a rigid frame,the method comprising: attaching a backing structure to the frame, thebacking structure comprising a planar region enclosed by the frame;forming a continuous layer of the first material over the planar regionand a portion of the frame surrounding the planar region; and releasingthe backing structure from the frame and the continuous layer of thefirst material to form the membrane.
 2. The method of claim 1, whereinthe planar region comprises a low-adhesion film, and wherein releasingthe backing structure comprises separating the continuous layer of thefirst material from the low-adhesion film.
 3. The method of claim 2,wherein the low-adhesion film comprises one of a non-stick surface, athermal-release tape, an ultra-violet release tape, and a sacrificiallayer.
 4. The method of claim 1, wherein the first material comprisesparylene, and wherein forming the continuous layer of the first materialcomprises vapor depositing parylene over the planar region and theportion of the frame surrounding the planar region.